COMPOSITION FOR DENTAL USE COMPRISING KRILL ENZYME

Abstract: Krill enzymes are used for the manufacture of a prophylactic composition for preventing dental plaque formation in a human subject, in particular for manufacture of a composition for decreasing the adhesive ability of plaque bacteria. A method for preventing dental plaque formation in a human subject and a method for decreasing the adhesive ability of plaque bacteria comprise administering such composition in the oral cavity of said subject.

Description: COMPOSITION FOR DENTAL USE COMPRISING KRILL ENZYME Technical Field Regular oral hygiene such as tooth brushing iε normally insufficient to provide adequate control of dental microbial plaque. Consequently, the incorporation of antimicrobial and plaque dissolving agents in dental products such as denti¬ frices has been proposed by dental researchers for a long time (CF. review by P.D. Marsh, J. Clin. Periodont. , 18, 462-67 (1991) . Apart from certain fluorides most compounds of potential use as antiplaque agents have turned out to be incompatible with either vehicle substances employed or the oral environment. Furthermore, recent research demonstrates that antiplaque agents should be designed to control rather than eliminate the dental plaque microflora (P.D. Marsh, Brit. Dent. J. 9, 174-77 (1991) . Like other parts of the digestive tract the oral cavity possesses a natural microflora which is bene¬ ficial to the host. Changes in the natural microflora may result in caries and periodontal disease. A number of antibacterial and surface active substances have been evaluated and tested for plaque control (Table 1) . Bisquanides and phenols (Chlorohexidine and Triclosan) have proved to be most effective. However, although showing a strong antibacterial action, they have undesirable short-term effects. Furthermore, possible long-term effects are causing concern. Recently, surface active agents such as amino alcohols have been introduced with the purpose of affecting the adhesion of bacteria to oral surfaces. Again, little is known about possible adverse long-term effects. Furthermore, bacteria are known to develop resistance to chemotherapeutic agents on prolonged use. Table 1: Classes and examples of antimicrobial antiplaque agents proposed for use in dentifrices. Class of inhibitor Some examples Antibiotics Tetracycline, penicillin Bisbiguanidines Chlorhexidine, alexidine Enzymes Mutanase, amyloglucosidase/ glucose oxidase Essential oils Thymol, eucalyptol Fluoride Monofluorophosphate Metal ions Zinc, stannous copper Plant extracts Sanguinarine Phenols Triclosan Quaternary ammonium compounds Cetylpyridinum chloride Surfactants Sodium lauryl, sulphate Several enzymes have been employed as antiplaque agents. However, in most cases it has not been possible to confirm promising laboratory tests in clinical trials. Proteases, mutanases and dextranases have been used primarily to dis¬ solve the plaque matrix which allows bacteria to adhere to tooth surface. An enzyme dentifrice which is presently being marketed (Zendium) contains an aminoglycosidase, the effect of which is based on an indirect antibacterial action. How- ever, the clinical effects seems to be too limited. The enzyme mixture from the digestive tract of the krill schrimp, Euphuasia superba, has proved to be unique in compo¬ sition and amazingly potent in action when used on organic substrates as compared to previously known digestive enzymes. Thus, a method of therapeutic an non-therapeutic cleaning is described in EP-B1-0, 107,634. Disclosure of Invention The invention concerns the use of these krill enzymes (KE) to prevent dental plaque on natural and artificial teeth, dental appliances, implants and soft tissues of the mouth. By apply¬ ing krill enzymes to these surfaces plaque formation, caries, yeast-cell infections and soft-tissue inflammation will be prevented. Compositions of enzymes from Antarctic krill (Euphausia superba) have been studied as potential oral prophylactic antiplaque/agents. A number of in vitro/in vivo studies have demonstrated that krill enzymes: retard plaque accumulation both in vitro and in vivo more effectively than Alcalase ® , do not exhibit an antibacterial effect and show mild antifungal properties against Candida albicans, common in oral opportunistic infections. It is concluded that krill enzymes offer highly promising properties as an antiplaque agent due to the high efficiency of krill enzymes in dissolving plaques and apparent biocompa- tibility. Oral diseases such aε caries, gingivitis/periodontitis and candidiasis are caused when microorganisms of the natural oral microflora accumulate on retentive surfaces such aε teeth and artifical reεtorations, thereby forming dental plaque (1,2,3). Poor or inadequate oral hygiene causes this plaque formation. Mechanical toothbrushing iε εtill the moεt efficient way to remove dental plaque (4, 15). Aε dental plaque consist of oral bacteria/microbes which aggregate on dental/oral surfaces by adherence, growth and production of extracellular organic matrix (18) , enzymes for debriding this organic debris have for a long time been con¬ sidered an ideal dental plaque remover (4) . Peptidase, pancr- eaεe, dextranaεeε and Alcalase ® (12) are all different enzymes tried over the years as dental plaque debriders with limited clinical success, the main reason for failure being weaknesε in catalyεt action and in retaining adequate and εufficient enzyme concentrations on substrates in the saliva- rich oral environment (4) . The concept of chemical plaque control has consequently been concentrated on antibacterial substanceε εuch aε chlor- hexidine (4, 16) and surface active agentε (21). These sub¬ stanceε, though, have some adverse side effects and the long term effect on the organism is uncertain. (19) . When enzymes are employed to remove plaque or to facilitate mechanical removal by toothbrushing they will merely have a limited effect on the inflammatory reactionε in the gingiva 1 εoft tiεεue (1, 3) εince there is strong evidence that food debris has its strongest devastating effect on teeth in the earliest phase after food consumption. The adherence of yeast-cellε (Candida albicanε) to dentureε and oral εoft tiεεue cauεes inflammatory reactions which are difficult to cure. (2) . The main reason iε, again, poor denture hygiene (5,6). Enzyme-containing denture-cleanerε are already on the market Alcalaεe ® being the more prominent (8, 9, 10, 12). Aε shown in the examples of this application, krill enzymeε have proved effective in prevention of dental plaque and yeaεt-cell adherence affecting adherence and metaboliεm of oral microorganiεmε and further improving the reduction of underlying inflammationε in εoft tiεεues. The proteolytic activity krill enzymes is often quantified in caεein units. In a krill enzyme mixture, the proteolytic activity is meaεured aε a part of the total enzymatic activity, which may comprise carbohydrate-splitting and lipolytic activity Example 1 Preparation of the enzyme composition Krill enzymes were extracted from the raw material, krill (Euphausia superba) according to standard procedureε described in (EP-A 107,634, EP-A 177,605). In short, krill waε frozen immediately after caught and εtored at -30°C before preparation. The thawed krill waε mixed with water, homogeniεed and centrifuged in the cold. The εupernatant was decanted and saved. Lipids were removed by extraction with organic εolvents, the water phase concentrated and freeze- dried thus resulting in a "crude krill enzyme preparation" suitable for dental use. Exampel 2 Detachement of yeast cells adhered to acrylic surfaces In accordance with a Japanese εtudy deεign (21) , εtrainε of Candida albicanε were incubated in 4 acrylic beakerε for 24 h at 37°C. The yeaεt cell then adhered to the acrylic surfaces which could be seen as a matt coating in the beakerε. Thiε model reεembleε yeast-cell infections on dental protheses. Solutions of krill enzymes (1 resp 2%) were added to two beakers while two other beakers served aε controls. 1 h later stereo microεcopy εhowed that >90% of countable yeaεt cells had detached from acrylic surfaces of krill enzyme treated beakers compared to controls. The results were photodocumen- ted (Table 1) Example 3 Retardation of plaque formation in dogs Two healthy beagle dogs were uεed in thiε study. In thiε split-mouth design, teeth on the control side were treated with a water solution applied 2 times daily for 2 minutes at 6 h intervals. Teeth on the active side were treated in the same way with krill enzymes in a 2% buffer solution. Baseline status of the teeth was gingival health (GI=0) and no plaque (PLI=0) obtained by thorough cleaning twice daily for two weekε prior to teεt εtart. The teεt run lasted for 3 weeks during which time the dogs were fed a carbohydrate-rich diet. The dogs were briefly sedated at treatment and registration. Gingival index according to Lδe and plaque index according to Silness & Lδe were used for regiεtration of plaque and gingi¬ val inflammation. Plaque on teeth waε made viεible with eosin colorin (Diaplac) . Teeth no P3 P4 and canines were used for regiεtration. In addition to clinical registration all regi- εtrationε were photodocumented. From the photos the amount of plaque on teeth buccal and approximal surfaces was asεessed. The amount of plaque was lesε on active εide compared to control teeth. Some of theεe results were statistically significant at p< 0.05 using the sign test. Example 4 Retardation of plaque formation in humans A test εubject εtarted with clinically clean and healthy teeth and gingiva (GI=0 & PLI=0) restrained from all oral hygiene for one week. During this time a water and a krill enzyme 2% buffer solution waε applied to control and active sides, respectively, in a split mouth design 3 timeε daily for 2 minutes. At the end of test period the result was photodocumented after that plaque on teeth had been visual¬ ized with eosin color (Diaplac) . There waε a noticable difference in plaque accumulation and aεεociated gingival inflammation between control and teεt εideε. Example 5 A dental paεte or gel iε prepared with the following compoεi- tion: Sodium monofluorophoεphate, dicalcium phoεphate dihydrate, giyceroi, sorbitol, sodium citrate, εodium lauryl sulphate, aromatic flavour, sodium εaccharinate, ethyl- hydroxy-4-benzoate, krill enzyme extract, citric acid colour¬ ing agents. Example 6 A chewing gum is prepared with the following composition: Chewing gum base, εodium fluoride, krill enzyme extract. Example 7 A mouth waεh and proεtheεiε cleaner iε prepared with the following compoεition: Baεe, carrier, εaline water εolution, krill enzyme extract. References 1. Lδe, H, Theilade, e & Jenεen, S.B. (1965) Experimental gingivitiε in man. J. of Periodontology 36, 177-1887. 2. Budts-Jδrgenεen, E. (1974) The significance of Candida albicans in denture stomatitiε. Scand J Dent Reε 82 151. 3. Socranεky, S.S. (1970) Relationship of bacteria to the aethiology of periodontal disease. J of Dent Res 49, 223-228. 4. Hull, P.S. (1980) Chemical inhibition of plaque. J of Clin Periodontology 7, 431-442. 5. Budtz-Jδrgensen, E. , Theilade, E, & Theilade, J. (1983) Quantitative relationεhip between yeasts and bacteria in denture-induced stomatitiε. Scand J Dent Res 91, 134. 6. Arendorf, T, Walker, D.M. (1979) Oral candidal populationε in health and diεeaεe. Br Dent J 147 267. 7. Connor, J.N.E., Schoenfels, CM., & Taylor, R.L. (1977) An evaluation of an enzyme denture cleanser. J Prosthet Dent 37, 147. 8. Budtz-Jδrgenεen, E. (1979) Material & methodε for clean¬ sing dentureε. J Proεthet Dent 42, 619. 10. Budtε-Jδrgenεen, E & Kelstrup, J. (1977) Enzymes as denture cleansers. Scan J Dent Reε 85, 209. 11. Axelεεon, P, & Lindhe, J. (1974) The effect a preventive programme on dental plaque, gingivitiε and carieε in εchool- children J of Clin Periodontology 1, 126-138. 12. Budtz-Jδrgensen, E, Kelεtrup, J & Poulεen, S. (1983) Reduction of formation of denture plaque by a protease (Alcalase) . Acta Odontol Scand 41, 93-98 13. Lang, N.P. Cumming, B.R. & Lδe, H. (1973) Toothbrush frequency is it iε related to plaque development and gingival health. J of Periodontology 44, 396-405. 14. Kornman, K.S., (1986) The role of supragingival plaque in the prevention and treatment of periodontal diseaεeε .. Reveiw. J of Periodont Reε 21, Suppl., 5-22. 15. Axelsson, P, Lindhe, J & Nystrδm, B, (1991) On the pre¬ vention of caries and periodontal disease. Results of a 15- year longitudinal study in adults. J of Clin Periodontology 18. 182-189. 16. Marsh, P.D. (1991) Dentifriceε containing new agents for the control of plaque. J Clin Periodontaol 18, 462-467. 17. Lδvdal, A et al., Combined effect of subgingival εcaling and controlled oral hygiene in the incidence og gingivitiε. Acta Odontol Scand 19, 537-555 (1961). 18. Krembel, J et al, (1969) Fractionation of human dental plaqueε. Arch oral Bio 14, 563-565. 19. Flϋtra, L Gjarmo P et al (1971) Side effects of chlor- hexidrive mouthwasheε Scand J of Dent Reε 79 119-125. 20. Collaert, Bruno (1992) Chemotherapeutic supragingival plaque control with delmopinol hydrochloride. Thesiε. 21. Shogo Minagi et al. (1987) Objective teεting of the efficiency of denture-cleanεing agentε. J of Proεthetic Dentistry 58 (5), 595-598.

Claims: CLAIMS 1. Use of krill enzyme for the manufacture of a prophylactic composition for preventing dental plaque formation in a human subject. 2. Use aε claimed in claim 1, wherein the compoεition com¬ prises 0.01 to 100 units of krill enzyme per 100 g of compo¬ sition. 3. Uεe aε claimed in claim 1 or 2, for manufacture of a compoεition for decreasing the adhesive ability of plaque bacteria. 4. A prophylactic composition for preventing dental plaque formation in a human subject comprising a prophylactically effective amount of krill enzyme in a carrier adapted for prophylactic treatment. 5. A prophylactic composition aε claimed in claim 4, compris¬ ing 0.01 to 100 units of krill enzyme per 100 g of composi¬ tion. 6. A prophylactic composition as claimed in claim 4 or 5 for decreasing the adheεive ability of plaque bacteria. 7. A method for preventing dental plaque formation in a human subject comprising administering in the oral cavity of said subject a prophylactically effective amount of krill enzyme in a carrier adapted for prophylactic treatment. 8. A method as claimed in claim 7, wherein the amount admin¬ istered iε 1 to 3 g, twice daily. 9. A method as claimed in claim 7 or 8, wherein a composition comprising 0.01 to 100 units of krill enzyme per 100 g of composition is administered. 10. A method for decreasing the adhesive ability of plaque bacteria in a human subject comprising administering in the oral cavity of said subject an effective amount of krill enzyme in a in a pharmaceutically acceptable carrier.

Inventors: HELLGREN KRISTIAN, HELLGREN LARS, MOHR VIGGO, VINCENT JAN, HELLGREN, KRISTIAN, HELLGREN, LARS, MOHR, VIGGO, VINCENT, JAN
Applicants: HELLGREN KRISTIAN, HELLGREN LARS, MOHR VIGGO, VINCENT JAN, HELLGREN, KRISTIAN, HELLGREN, LARS, MOHR, VIGGO, VINCENT, JAN
Application year: 1994
Main classification: A61K35
IPC codes: A23G4, A61K8, A61K35, A61K38, A61P1, A61P31, A61Q11